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李庆霞,王娟,赵文清,刘家云,黄红艳,张明,杨安钢.RNA干扰survivin基因增强SKBr-3细胞对多柔吡星敏感性的体外研究[J].陆军军医大学学报(原第三军医大学学报),2010,32(11):1158-1161.
Li Qingxia,Wang Juan,Zhao Wenqing,Liu Jiayun,Huang Hongyan,Zhang Ming,Yang Angang.RNAinterfered survivin gene increases sensitivity of SKBr-3 cells to doxorubicin: an in vitro study[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(11):1158-1161.
李庆霞,王娟,赵文清,刘家云,黄红艳,张明,杨安钢.RNA干扰survivin基因增强SKBr-3细胞对多柔吡星敏感性的体外研究[J].陆军军医大学学报(原第三军医大学学报),2010,32(11):1158-1161. DOI:
Li Qingxia,Wang Juan,Zhao Wenqing,Liu Jiayun,Huang Hongyan,Zhang Ming,Yang Angang.RNAinterfered survivin gene increases sensitivity of SKBr-3 cells to doxorubicin: an in vitro study[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(11):1158-1161. DOI:
目的 探讨抑制survivin基因的表达对人乳腺癌SKBr-3细胞化疗敏感性的影响。 方法 构建针对survivin基因序列特异性shRNA的表达载体,脂质体转染SKBr-3,G418筛选阳性克隆。实验分为RNA干扰组(S1P),脂质体对照组(H1P)和空白对照组。光镜观察转染后细胞的形态变化;0.5 μg/ml 多柔吡星作用于转染后的细胞,电镜观察细胞形态学变化;TUNEL染色、Annexin V荧光标记检测细胞凋亡,MTT法检测细胞增殖的抑制率。 结果 成功构建具有新霉素抗性的靶向survivin的序列特异性shRNA表达载体,基因测序完全正确;转染后干扰组细胞均发生细胞凋亡的形态学改变;低剂量多柔吡星诱导后,TUNEL染色结果,S1P组凋亡细胞比例(23.6±4.8)%明显高于H1P组(4.1±1.1) %和空白对照组(3.2±1.4) %(P0.05),表明转染了RNA干涉载体的SKBr-3细胞对低剂量多柔吡星的敏感性显著增加;Annexin-V染色结果:空白对照组凋亡比例为(17.43±3.12)%,H1P组为(20.51±4.67)%,S1P组为(68.76±8.49)%,后者与前两者比较均有统计学差异(P0.05)。干扰组细胞凋亡率、细胞增殖的抑制率均明显增加,差异有统计学意义(P0.05)。 结论 体外实验证实抑制survivin基因的表达可提高乳腺癌SKBr-3细胞对多柔吡星的敏感性。
Objective To study the effect of RNA-interfered survivin gene on chemosensitivity of breast cancer cell line SKBr-3 to doxorubicin. Methods A shRNA expression vector
ΔpcDNA3-H1-S1
was constructed according to the specificity of survivin gene. SKBr-3 cells were transfected with liposomeencapsulated plasmids and positive clones were screened with G418. SKBr-3 cells were divided into RNA interference group
liposome control group and blank control group. Change of morphology in cells transfected with 0.5 μg/ml doxorubicin was observed under an optical or electron microscope. Cell apoptosis was detected by TUNEL assay with Annexin-Ⅴ fluorescence staining
and inhibiting rate of cell proliferation was detected by MTT assay. Results The survivin gene sequence specific shRNA expression vector resistant to neomycin was successfully constructed
which was confirmed by DNA sequencing. The morphologic change of apoptotic cells was observed in RNA interference group. The rate of apoptotic cells was significantly higher in RNA interference group than in liposome control group and blank control group [(23.6±4.8)% vs (4.1±1.1)% and (3.2±1.4)%] after TUNEL staining (P0.05)
indicating that the sensitivity of SKBr-3 cells to doxorubicin was increased in RNA-interfered vector. The rate of apoptotic cells was significantly lower in liposome control group and blank control group than in RNA interference group [(17.43±3.12)% and (20.51±4.67)% vs (68.76±8.49)%] after Annexin-V staining (P0.05). Conclusion The chemosensitivity of SKBr-3 cells to doxorubicin can be significantly increased by inhibiting the expression of survivin gene with RNA interference technology.
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